Selective Microbial Genomic DNA Isolation Using Restriction Endonucleases
| Autor: | Kimberly T. Helzer, Helen E. Barnes, Dan Sphar, Christopher Q. Weston, R. Allyn Forsyth, Laura Day, Robert T. Yamamoto, Shannon Waltz, Guohong Liu, Paula King, Long K. Pham |
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| Rok vydání: | 2014 |
| Předmět: |
DNA
Bacterial Molecular biology Science Genomics Microbial Genomics Biology Biomolecular isolation Microbiology DNA sequencing Microbial Ecology chemistry.chemical_compound Genetics Escherichia coli Genomic library Genome Sequencing Sequencing Techniques Gram Negative Bacteria Multidisciplinary Biology and life sciences Ecology Bacterial Genomics High-Throughput Nucleotide Sequencing Bacteriology DNA Restriction Enzymes Bacterial Genomes DNA isolation DNA extraction genomic DNA Restriction enzyme Molecular biology techniques chemistry Biochemistry Medical Microbiology Metagenomics Medicine Microbiome Genome Bacterial DNA Research Article |
| Zdroj: | PLoS ONE PLoS ONE, Vol 9, Iss 10, p e109061 (2014) |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0109061 |
| Popis: | To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment. |
| Databáze: | OpenAIRE |
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